Stabilization of pet operon plasmids and ethanol production in Escherichia coli strains lacking lactate dehydrogenase and pyruvate formate-lyase activities.

In the last decade, a major goal of research in biofuels has been to metabolically engineer microorganisms to ferment multiple sugars from biomass or agricultural wastes to fuel ethanol. Escherichia coli strains genetically engineered to contain the pet operon (Zymomonas mobilis pyruvate decarboxylase and alcohol dehydrogenase B genes) produce high levels of ethanol. Strains carrying the pet operon in plasmid (e.g., E. coli B/pLOI297) or in chromosomal (e.g., E. coli KO11) sites require antibiotics in the media to maintain genetic stability and high ethanol productivity. To overcome this requirement, we used the conditionally lethal E. coli strain FMJ39, which carries mutations for lactate dehydrogenase and pyruvate formate lyase and grows aerobically but is incapable of anaerobic growth unless these mutations are complemented. E. coli FBR1 and FBR2 were created by transforming E. coli FMJ39 with the pet operon plasmids pLOI295 and pLOI297, respectively. Both strains were capable of anaerobic growth and displayed no apparent pet plasmid losses after 60 generations in serially transferred (nine times) anaerobic batch cultures. In contrast, similar aerobic cultures rapidly lost plasmids. In high-cell-density batch fermentations, 3.8% (wt/vol) ethanol (strain FBR1) and 4.4% (wt/vol) ethanol (strain FBR2) were made from 10% glucose. Anaerobic, glucose-limited continuous cultures of strain FBR2 grown for 20 days (51 generations; 23 with tetracycline and then 28 after tetracycline removal) showed no loss of antibiotic resistance. Anaerobic, serially transferred batch cultures and high-density fermentations were inoculated with cells taken at 57 generations from the previous continuous culture. Both cultures continued to produce high levels of ethanol in the absence of tetracycline. The genetic stability conferred by selective pressure for pet-containing cells without requirement for antibiotics suggests potential commercial suitability for E. coli FBR1 and FBR2.     

Fermentation of corn fibre sugars by an engineered xylose utilizing Saccharomyces yeast strain

The ability of a recombinant Saccharomyces yeast strain to ferment the sugars glucose, xylose, arabinose and galactose which are the predominant monosaccharides found in corn fibre hydrolysates has been examined. Saccharomyces strain 1400 (pLNH32) was genetically engineered to ferment xylose by expressing genes encoding a xylose reductase, a xylitol dehydrogenase and a xylulose kinase. The recombinant efficiently fermented xylose alone or in the presence of glucose. Xylose-grown cultures had very little difference in xylitol accumulation, with only 4 to 5g/l accumulating, in aerobic, micro-aerated and anaerobic conditions. Highest production of ethanol with all sugars was achieved under anaerobic conditions. From a mixture of glucose (80g/l) and xylose (40g/l), this strain produced 52g/l ethanol, equivalent to 85% of theoretical yield, in less than 24h. Using a mixture of glucose (31g/l), xylose (15.2g/l), arabinose (10.5g/l) and galactose (2g/l), all of the sugars except arabinose were consumed in 24h with an accumulation of 22g ethanol/l, a 90% yield (excluding the arabinose in the calculation since it is not fermented). Approximately 98% theoretical yield, or 21g ethanol/l, was achieved using an enzymatic hydrolysate of ammonia fibre exploded corn fibre containing an estimated 47.0g mixed sugars/l. In all mixed sugar fermentations, less than 25% arabinose was consumed and converted into arabitol.     

High-resolution autoradiography of nuclear modifications during and after heat treatment ofNeurospora crassa

Summary The appearance of perinucleolar electron-dense spots in the nuclei of macroconidia ofNeurospora crassa incubated at 46C and their disaggregation after shift-down to 25 C have been investigated by high-resolution autoradiography after (5-³H) uridine pulses with or without chase periods. The RNA of these ribonucleoprotein-rich dense spots has been found to originate mainly from the heatsensitive nucleolus; after return to 25 C, the nucleolar activity was recovered and the RNA material stored either in an unprocessed or a mature rRNA form in the dense spots was found to be progressively extruded into the cytoplasm.     

Identification of tryptophan pyrrolase in liver lysosomes after treatment of rats with hydrocortisone and chloroquine

Liver lysosomes isolated from rats treated with hydrocortisone and chloroquine were found to contain an increased amount of protein including 3% of the tryptophan pyrrolase enzyme activity and 5% of the antigenic activity. Immunoprecipitates were obtained by incubating lysosomes with antibody to tryptophan pyrrolase. Analysis of these immunoprecipitates by sodium dodecyl sulfate polyacrylamide gel electrophoresis revealed tryptophan pyrrolase subunits (MW 45,000) and smaller polypeptides, presumably proteolytic degradation products of intact subunits.     

Effect of Urea and Thiourea on Migration Activity of Climbing Perch Anabas testudineus

Journal of Ichthyology - The influence of urea and thiourea on the dynamics of the migration activity of climbing perch Anabas testudineus was studied. The exposure of climbing perch to 0.05%...     

Isoswinholide B and swinholide K,potently cytotoxic dimeric macrolides from Theonella swinhoei

Chemical investigation of an Indonesian specimen of Theonella swinhoei afforded the new dimeric macrolides isoswinholide B (5) and swinholide K (6), along with the known swinholides A (1), B (2) and D (3) and isoswinholide A (4). Isoswinholide B showed an unprecedented 21/19′ lactonization pattern, while swinholide K included an sp² methylene attached at C-4 and an additional oxymethine group at C-5, whose configuration has been determined through application of J-based configuration analysis. The isolated swinholides (1–6), with the exception of isoswinholide B, showed a cytotoxic activity on HepG2 (hepatocarcinoma cell line) in the nanomolar range.     

Use of tropical maize for bioethanol production

Tropical maize is an alternative energy crop being considered as a feedstock for bioethanol production in the North Central and Midwest United States. Tropical maize is advantageous because it produces large amounts of soluble sugars in its stalks, creates a large amount of biomass, and requires lower inputs (e.g. nitrogen) than grain corn. Soluble sugars, including sucrose, glucose and fructose were extracted by pressing the stalks at dough stage (R4). The initial extracted syrup fermented faster than the control culture grown on a yeast extract/phosphate/sucrose medium. The syrup was subsequently concentrated 1.25–2.25 times, supplemented with urea, and fermented using Saccharomyces cerevisiae for up to 96 h. The final ethanol concentrations obtained were 8.1 % (v/v) to 15.6 % (v/v), equivalent to 90.3–92.2 % of the theoretical yields. However, fermentation productivity decreased with sugar concentration, suggesting that the yeast might be osmotically stressed at the increased sugar concentrations. These results provide in-depth information for utilizing tropical maize syrup for bioethanol production that will help in tropical maize breeding and development for use as another feedstock for the biofuel industry.     

Cellulosic Butanol (ABE) Biofuel Production from Sweet Sorghum Bagasse (SSB): Impact of Hot Water Pretreatment and Solid Loadings on Fermentation Employing <Emphasis Type="Italic">Clostridium beijerinckii</Emphasis> P260

A novel butanol fermentation process was developed in which sweet sorghum bagasse (SSB) was pretreated using liquid hot water (LHW) pretreatment technique followed by enzymatic hydrolysis and butanol (acetone butanol ethanol (ABE)) fermentation. A pretreatment temperature of 200 °C resulted in the generation of a hydrolyzate that inhibited butanol fermentation. When SSB pretreatment temperature was decreased to 190 °C (0-min holding time), the hydrolyzate was successfully fermented without inhibition and an ABE productivity of 0.51 g L^?1 h^?1 was achieved which is comparable to the 0.49 g L^?1 h^?1 observed in the control fermentation where glucose was used as a feedstock. These results are based on the use of 86 g L^?1 SSB solid loadings in the pretreatment reactors. We were also able to increase SSB solid loadings from 120 to 200 g L^?1 in the pretreatment step (190 °C) followed by hydrolysis and butanol fermentation. As pretreatment solid loadings increased, ABE yield remained in the range of 0.38–0.46. In these studies, a maximum ABE concentration of 16.88 g L^?1 was achieved. Using the LHW pretreatment technique, 88.40–96.00 % of polymeric sugars (cellulose + hemicellulose) were released in the SSB hydrolyzate. The LHW pretreatment technique does not require chemical additions and is environmentally friendly, and the hydrolyzate can be used successfully for butanol fermentation.     

Sortase-catalysed anchoring of surface proteins to the cell wall of Staphylococcus aureus

Many surface proteins of Gram-positive bacteria are anchored to the cell wall envelope by a transpeptidation mechanism, requiring a C-terminal sorting signal with a conserved LPXTG motif. Sortase, a membrane protein of Staphylococcus aureus, cleaves polypeptides between the threonine and the glycine of the LPXTG motif and catalyses the formation of an amide bond between the carboxyl-group of threonine and the amino-group of peptidoglycan cross-bridges. S. aureus mutants lacking the srtA gene fail to anchor and display some surface proteins and are impaired in the ability to cause animal infections. Sortase acts on surface proteins that are initiated into the secretion (Sec) pathway and have their signal peptide removed by signal peptidase. The S. aureus genome encodes two sets of sortase and secretion genes. It is conceivable that S. aureus has evolved more than one pathway for the transport of 20 surface proteins to the cell wall envelope.     

Anchoring of surface proteins to the cell wall of Staphylococcus aureus. III. Lipid II is an in vivo peptidoglycan substrate for sortase-catalyzed surface protein anchoring

Surface proteins of Staphylococcus aureus are anchored to the cell wall peptidoglycan by a mechanism requiring a C-terminal sorting signal with an LPXTG motif. Surface proteins are first synthesized in the bacterial cytoplasm and then transported across the cytoplasmic membrane. Cleavage of the N-terminal signal peptide of the cytoplasmic surface protein P1 precursor generates the extracellular P2 species, which is the substrate for the cell wall anchoring reaction. Sortase, a membrane-anchored transpeptidase, cleaves P2 between the threonine (T) and the glycine (G) of the LPXTG motif and catalyzes the formation of an amide bond between the carboxyl group of threonine and the amino group of cell wall cross-bridges. We have used metabolic labeling of staphylococcal cultures with [(32)P]phosphoric acid to reveal a P3 intermediate. The (32)P-label of immunoprecipitated surface protein is removed by treatment with lysostaphin, a glycyl-glycine endopeptidase that separates the cell wall anchor structure. Furthermore, the appearance of P3 is prevented in the absence of sortase or by the inhibition of cell wall synthesis. (32)P-Labeled cell wall anchor species bind to nisin, an antibiotic that is known to form a complex with lipid II. Thus, it appears that the P3 intermediate represents surface protein linked to the lipid II peptidoglycan precursor. The data support a model whereby lipid II-linked polypeptides are incorporated into the growing peptidoglycan via the transpeptidation and transglycosylation reactions of cell wall synthesis, generating mature cell wall-linked surface protein.